An assay for RNA oxidation induced abasic sites using the Aldehyde Reactive Probe

Free Radic Res. 2011 Feb;45(2):237-47. doi: 10.3109/10715762.2010.535529. Epub 2010 Nov 10.

Abstract

There have been several reports describing elevation of oxidized RNA in ageing or age-related diseases, however RNA oxidation has been assessed solely based on 8-hydroxy-guanosine levels. In this study, Aldehyde Reactive Probe (ARP), which was originally developed to detect DNA abasic sites, was used to assess RNA oxidation. It was found that ARP reacted with depurinated tRNA(Phe) or chemically synthesized RNA containing abasic sites quantitatively to as little as 10 fmoles, indicating that abasic RNA is recognized by ARP. RNA oxidized by Fenton-type reactions, γ-irradiation or peroxynitrite increased ARP reactivity dose-dependently, indicating that ARP is capable of monitoring oxidized RNA mediated by reactive oxygen species or reactive nitrogen species. Furthermore, oxidative stress increased levels of ARP reactive RNA in cultured cells. These results indicate the versatility of the assay method for biologically relevant oxidation of RNA. Thus, this study developed a sensitive assay for analysis of oxidized RNA.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aging
  • Apurinic Acid / chemistry
  • Biological Assay / methods*
  • Biotin / analogs & derivatives*
  • Biotin / chemistry
  • Biotin / metabolism
  • Female
  • Gamma Rays
  • Guanosine / analogs & derivatives
  • Guanosine / chemistry
  • Guanosine / metabolism
  • HeLa Cells
  • Humans
  • Oxidation-Reduction / drug effects
  • Oxidation-Reduction / radiation effects
  • Peroxynitrous Acid / pharmacology
  • Polynucleotides / chemistry
  • Quantitative Structure-Activity Relationship
  • RNA / chemistry
  • RNA / metabolism*
  • RNA / radiation effects
  • Reactive Nitrogen Species / metabolism
  • Reactive Oxygen Species / metabolism

Substances

  • Polynucleotides
  • Reactive Nitrogen Species
  • Reactive Oxygen Species
  • apyrimidinic acid
  • Apurinic Acid
  • Guanosine
  • O-(biotinylcarbazoylmethyl)hydroxylamine
  • Peroxynitrous Acid
  • 8-hydroxyguanosine
  • RNA
  • Biotin